1. Field of the Invention
This invention relates to a cover slip for use in a microscope, suitable for observing a biological sample by use of a water-immersed objective lens, in particular, with a high power and a high numerical aperture.
2. Description of the Related Art
Recently, three-dimensional structural analysis of a cytoskeleton or a chromosome by use of a laser scanning microscope (LSM) with high contrast and resolution, or locally detection of a fluorescent substance in a cell by use of a fluorescent antibody method using an LSM, have attracted great public attention as regards basic research of biological and medical. The greatest merit of the LSM is that a sample is optically sliced by a confocal optical system so as to obtain a three-dimensional image. When optical slicing is performed, not only a surface portion but also an interior portion of the sample must be observed. Hence, observation of the sample is carried out through a cover slip and the sample itself in the case of a standard microscope, and often through a thin layer of culture solution in the case of an inverted microscope.
Observation of the interior of a sample (e.g. a cell) through a culture solution by use of a conventional oil-immersed objective lens can considerably deteriorate the focusing accuracy, since the refractive index of the sample and the culture solution are substantially equal to that (1.33) of water and greatly differs from the refractive index (1.515) of the immersion oil. In fact, when the optical slicing is performed using an oil-immersed objective lens with a numerical aperture of approx. 1.4, a good image cannot be obtained from a portion of the sample located deeper than about 30 .mu.m below its surface. On the other hand, a water-immersed objective lens having a refractive index which does not greatly differ from that of the sample and the culture solution is used, the deterioration of the focusing accuracy is small, so that a good image of the optical slicing from a portion located as deep as possible within the working Distance (WD) can be obtained.
However, the material of the conventional cover slip is selected to have a refractive index (1.521) according to the refractive index of the immersion oil. Accordingly, if the cover slip is used in combination with the water-immersed objective lens, an error in the thickness of the cover slip will deteriorate the focusing accuracy of the objective lens because of the difference of refractive index (0.191) between the cover slip and water of the objective lens. An error in the thickness of the cover slip is generally about .+-.0.02 mm based on current manufacturing techniques and costs. This amount of error greatly deteriorates the focusing accuracy of the objective lens which has a high power and a high numerical aperture.
Published Unexamined Japanese Patent Application No. 56-50312 and German Patent No. 2655041 disclose techniques developed in light of the above-described disadvantage. The Japanese application discloses use of an acrylate resin as the material of a cover slip for use in a microscope. The acrylate resin has a refractive index of approx. 1.5, which is still much greater than that of water, and hence has the same defect as the material used to form a standard cover slip. On the other hand, the German patent discloses a technique for displacing, along the optical axis, a particular lens component in a microscope objective lens in accordance with an error in the thickness of the cover slip, so as to compensate for deterioration in focusing accuracy. Use of this technique, however, makes it difficult to design the objective lens, and to adjust the lens position to one at which deterioration in focusing accuracy is minimized.